Recently, several naturally occurring proteinaceous molecules have been identified based on their trophic activity on various types of neurons. These molecules are termed "neurotrophic factors". Neurotrophic factors are endogenous, soluble proteins that play a major role in neuronal survival and growth during development, as well as in the functional maintenance and plasticity of mature neurons; see Fallon and Laughlin, Neurotrophic Factors,Academic Press, San Diego, Calif. (1993). In view of their ability to promote neuron regeneration and to prevent neuron death and degeneration, it has been postulated that neurotrophic factors might be useful in treating neurodegenerative conditions of the nervous system, such as, for example, Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis and stroke.
Nerve damage is caused by conditions that compromise the survival and/or proper function of one or more types of nerve cells, including:
(1) physical injury, which causes the degeneration of the axonal processes (which in turn causes nerve cell death) and/or nerve cell bodies near the site of injury, (2) temporary or permanent cessation of blood flow (ischemia) to parts of the nervous system, as in stroke, (3) intentional or accidental exposure to neurotoxins, such as the cancer and AIDS chemotherapeutic agents cisplatinum and dideoxycytidine, respectively, (4) chronic metabolic diseases, such as diabetes or renal dysfunction, or (5) neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, and Amyotrophic Lateral Sclerosis, which result from the degeneration of specific neuronal populations. In order for a particular neurotrophic factor to be potentially useful in treating nerve damage, the class or classes of damaged nerve cells must be responsive to the factor; different neurotrophic factors typically affect distinctly different classes of nerve cells. It has been established that all neuron populations are not responsive to or equally affected by all neurotrophic factors.
The first neurotrophic factor to be identified was nerve growth factor (NGF). NGF is the first member of a defined family of trophic factors, called the neurotrophins, that currently includes brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), NT-4/5, and NT-6 (Thoenen, Trends. Neurosci., 14:165-170, 1991; Lapchak et al., Rev. Neurosci., 3:1-10, 1993; Bothwell, Ann. Rev. Neurosci., 18:223-253, 1995). These neurotrophins are known to act through the family of trk tyrosine kinase receptors, i.e., trkA, trkB, trkC, and the low affinity p75 receptor (Lapchak et al., Rev. Neurosci., 3:1-10, 1993; Bothwell, Ann. Rev. Neurosci., 18:223-253, 1995; Chao et al., TINS, 18:321-326, 1995). In the central nervous system (CNS), the expression of trKA, the receptor for NGF, is almost exclusively limited to the cholinergic neurons in the basal forebrain (Venero et al., Neuroreport, 4:959-962, 1993), which also express p75 and trkB. These cholinergic neurons are of particular neurologic interest, because cholinergic neuronal degeneration and/or dystrophy is a hallmark of Alzheimer's disease (Hefti, J. Neurobiol., 25:1418-1435, 1994; Olson, Neurochem. Jul., 15:1-3, 1994). The basal forebrain cholinergic neurons can be readily identified in morphologic preparations using acetylcholinesterase histochemistry or with immunohistochemistry using antibody to choline acetyltransferase (ChAT), the synthetic enzyme for acetylcholine, or to p75 (Batchelor et al., J. Comp. Neurol., 284:187-204, 1989; Kiss et al., Neurosci., 27:731-748, 1988; Woolf et al., Neuroscience, 30:143-152, 1989).
Glial cell line-derived neurotrophic factor (GDNF) is a recently discovered protein identified and purified using assays based upon its efficacy in promoting the survival and stimulating the transmitter phenotype of mesencephalic dopaminergic neurons vitro (Lin et al., Science, 260:1130-1132, 1993). GDNF is a glycosylated, disulfide-bonded homodimer that is distantly related to the transforming growth factor-.beta. (TGF-.beta.) superfamily of neurotrophic proteins (Krieglstein et al., EMBO J., 14:736-742, 1995; Poulsen et al., Neuron ,13:1245-1252, 1994). GDNF has been cloned, and the recombinant human GDNF (rhuGDNF) exerts trophic and survival-promoting actions on substantia nigra dopaminergic neurons and spinal cord motor neurons in vitro, as well as in vivo (Beck et al., Nature, 273:339-341, 1995; Henderson et al., Science, 266:1130-1132, 1994; Tomac et al., Nature, 273: 335-339; Yan et al., Nature, 273: 341-343; Zurn et al., Neuroreport, 6:113-118, 1994). In vivo, treatment with exogenous GDNF stimulates the dopaminergic phenotype of substantia nigra neurons and restores functional deficits induced by axotomy or dopaminergic neurotoxins in animal models of Parkinson's disease, a neurodegenerative disease characterized by the loss of dopaminergic neurons (Hudson et al., Brain Res. Bull., 36:425-432, 1995; Hoffer et al., Neurosci Lett., 182:107-111, 1994). Although originally thought to be relatively specific for dopaminergic neurons, at least in vitro, subsequent experiments have found that GDNF has neurotrophic efficacy on brain stem and spinal cord cholinergic motor neurons, both in vivo and in vitro (Oppenheim et al., Nature, 373:344-346, 1995; Zurn et al., Neuroreport, 6:113-118, 1994; Yan et al., Nature, 373: 341-344, 1995; Henderson et al., Science, 266:1062-1064, 1994). GDNF is, therefore, a factor with potential therapeutic benefit in the treatment of degenerative disorders of spinal cord motor neurons, such as amyotrophic lateral sclerosis.
Thus, evidence is beginning to emerge indicating that GDNF may have a larger spectrum of neurotrophic targets besides mesencephalic dopaminergic and somatic motor neurons (Yan and Matheson, Nature, 373:341-344, 1995; Miller et al., Soc. Neurosci. Abstr., 20:1300, 1994). GDNF messenger RNA (mRNA) has been detected in muscle and Schwann cells in the peripheral nervous system and in type I astrocytes (Schaas et al., Exp. Neurol, 124:368-371, 1993) in the central nervous system. GDNF mRNA is also expressed in high levels in the developing rat striatum (Stromberg et al., Exp. Neurol., 124:401-412, 1993), and in low levels in regions of the adult rat and human central nervous system, including striatum, hippocampus, cortex and spinal cord (Springer et al., Exp. Neurol, 127:167-170, 1994).
Of general interest to the present invention is WO93/06116 (Lin et al., Syntex-Synergen Neuroscience Joint Venture), published Apr. 1, 1993, which reports that GDNF is useful for the treatment of nerve injury, including injury associated with Parkinson's Disease. Also of interest are a report in Schmidt-Kastner et al., Mol. Brain Res., 26:325-330, 1994 that GDNF mRNA became detectable and was upregulated after pilocarpine-induced seizures; reports in Schaar et al., Exp. Neurol, 124:368-371, 1993 and Schaar et al., Exp. Neurol., 130:387-393, 1994 that basal forebrain astrocytes expressed moderate levels of GDNF mRNA under culture conditions, but that GDNF did not alter basal forebrain ChAT activity; and a report in currently pending U.S. application Ser. No. 08/535,682 filed Sep. 28, 1995 that GDNF is useful for treating injury or degeneration of basal forebrain cholinergic neurons.
In mammals, a number of ophthalmic neurodegenerative conditions or diseases involve injury or degeneration of photoreceptors. Trophic factors capable of promoting the survival or regeneration of these neurons would provide useful therapies for the treatment of such diseases.
Photoreceptors are a specialized subset of retinal neurons, that are responsible for vision. Photoreceptors consist of rods and cones which are the photosensitive cells of the retina. Each rod and cone elaborates a specialized cilium, referred to as an outer segment, that houses the phototransduction machinery. The rods contain a specific light-absorbing visual pigment, rhodopsin. There are three classes of cones in humans, characterized by the expression of distinct visual pigments: the blue cone, green cone and red cone pigments. Each type of visual pigment protein is tuned to absorb light maximally at different wavelengths. The rod rhodopsin mediates scotopic vision (in dim light), whereas the cone pigments are responsible for photopic vision (in bright light). The red, blue and green pigments also form the basis of color vision in humans. The visual pigments in rods and cones respond to light and generate an action potential in the output cells, the rod bipolar neurons, which is then relayed by the retinal ganglion neurons to produce a visual stimulus in the visual cortex.
In humans, a number of diseases of the retina involve the progressive degeneration and eventual death of photoreceptors, leading inexorably to blindness. Degeneration of photoreceptors, such as by inherited retinal dystrophies (e.g., retinitis pigmentosa), age-related macular degeneration and other maculopathies, or retinal detachment, are all characterized by the progressive atrophy and loss of function of photoreceptor outer segments. In addition, death of photoreceptors or loss of photoreceptor function results in partial deafferentation of second order retinal neurons (rod bipolar cells and horizontal cells) in patients with retinal dystrophies, thereby decreasing the overall efficiency of the propagation of the electrical signal generated by photoreceptors. Trophic factors that are capable of rescuing photoreceptors from cell death and/or restoring the function of dysfunctional (atrophic or dystrophic) photoreceptors may represent useful therapies for the treatment of such conditions.
There is some evidence that certain protein factors may promote the survival of photoreceptors. For example, photoreceptors can be rescued to some extent by basic fibroblast growth factor (bFGF) in Royal College of Surgeons (RCS) rats and in albino rats that have been damaged by exposure to constant light (Faktorovich et al., Nature, 347:83-86, 1990). RCS rats have an inherited mutation of a gene expressed in the retinal pigment epithelium (RPE), that results in the failure of the RPE to phagocytize the continuously shed portions of the photoreceptor outer segments and causes photoreceptor degeneration and eventually cell death. A single injection of bFGF into the vitreous body or into the subretinal space, the extracellular space surrounding rods and cones, at the onset of the degeneration transiently rescues photoreceptors (Faktorovich et al., Nature, 347:83-86, 1990 ). In the light-damaged model in albino rats, bFGF injected into the subretinal space or the vitreous body two days prior to the onset of constant illumination significantly protects photoreceptors from light injury and prevents cell death (LaVail et al., Proc. Natl. Acad. Sci. USA, 89:11249-11253, 1992). In this model, photoreceptor survival was also seen with acidic FGF (aFGF), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), and interleukin-1.beta. (IL-1.beta.). Moderate effects were observed with neurotrophin-3 (NT-3), insulin-like-growth factor II (IGF-II) and tumor necrosis factor-alpha (TNF-alpha). Nerve growth factor (NGF), epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and IGF-I had no effect (LaVail et al., Proc. Natl. Acad. Sci. USA, 89:11249-11253, 1992). Also see WO 93/15608, LaVail et al.
Although bFGF is efficacious in the RCS rat and light-induced damage rat models, its therapeutic utility in humans is very limited, due to its hypotensive, mitogenic and potent angiogenic activities. In fact, bFGF injected into the vitreous body causes the invasion of blood-derived macrophages in the inner retina and can produce a massive proliferative vitreoretinopathy (Faktorovich et al., Nature, 347:83-86, 1990). It has also been determined, using polymerase chain reaction technology, that messenger RNA for GDNF is expressed in the eyes of postnatal day 6 and adult rats, essentially associated with the neural retina and the retinal pigment epithelium. The RPE cells produce, store and transport a variety of factors that are responsible for the survival and functional maintenance of photoreceptors. The RPE cells are also indispensable to the phototransduction process: they clear up by phagocytosis the shed tips of the outer segments of photoreceptors and recycle vitamin A. The transplantation of normal RPE cells into retinas of RCS rats prevents photoreceptor cell death (Li and Turner, Exp. Eye Res., 47:911-917, 1988; Mullen and LaVail, Science, 192:799-801, 1976), suggesting the production by RPE cells of a diffusable trophic factor for photoreceptors.
There continues to exist a need for methods and therapeutic compositions useful for the treatment of photoreceptor cell injury. Such methods and therapeutic compositions would ideally protect the photoreceptors from progressive injury and promote the survival or regeneration of the damaged neuron population, without severe side effects.